The long term objective of this project is to understand the structural basis for coupling ATP hydrolysis to proton transport by the plasma membrane H+-ATPase of yeast. Energy coupling is a fundamental process in biology that frequently involves the conversion of chemical energy to mechanical work. The mechanics underlying coupling in transport enzymes are poorly understood. the yeast H+-ATPase is a proton pump that has been chosen for these studies because it is amenable to biochemical and genetic analyses that are essential for probing coupling. In addition, the H+- ATPase shares significant sequence homology with, and closely resembles in structure and function, important P-type ATPases from animal cells including the Na+,K+-ATPase, H+, K+-ATPase and Ca2+- ATPase. This project will focus on a localized region of protein structure that was implicated from genetic studies, completed in the prior project period, to play an important role in coupling. This region consists of a cytoplasmic hydrophilic loop domain termed the 'phosphatase' domain and includes transmembrane segments 2 and 3. The operating premise in this proposal is that this region provides a structural linkage between proton translocation and ATP hydrolysis domains. This proposal seeks to probe this region genetically in an effort to provide detailed evidence for the role of specific amino acids and/or localized regions of protein structure in coupling. Localized random mutagenesis will be used to generate mutations within the target region and potential pmal coupling mutants will be selected on the basis of hygromycin B resistance and low pH sensitivity. The mutations will be genetically identified and mutant enzymes characterized for assembly and stability properties, the kinetics of ATP hydrolysis and proton transport, and the stoichiometry of H+ transported to ATP hydrolyzed (coupling ratio). Primary site mutations inducing prominent cellular and biochemical phenotypes will be used in revertant analyses to identify local and long-range protein structure interactions. Site-directed mutagenesis will be used to modify residues identified from initial screening routines to be important to function and amino acid residues flanking important primary sites will be extensively modified by saturation mutagenesis to examine effects of localized structure on coupling. Finally, molecular modeling will be used as a visualization and prediction tool to model local regions of protein structure, as well as interactions between closely apposed protein structure elements. The methodological approaches include random, saturation and site- directed mutagenesis, DNA sequence analysis, PCR cloning and mutagenesis, revertant analysis, ATP hydrolysis and H+-transport kinetics, SDS-PAGE, Western blot analysis, H+/ATP stoichiometry and molecular modeling.
Showing the most recent 10 out of 23 publications
